Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization
Abstract
:1. Introduction
2. Results
2.1. Phosphorylation of an RSV PCtail-Derived Peptide Increases Affinity for RSV NNTD
2.2. Fluorescence Polarization Reveals a Potential Secondary Binding Site on RSV NNTD for Fluorescein-Labeled RSV P11 Peptide
2.3. The Complex betweeen Fluorescein-Labeled C-Terminal P Peptides and Full-Length N Protein Provides a Robust Model of the RSV N-P Interaction for FP Measurements
2.4. Measurement of the Binding Affinity of RSV Fluorescein-P Peptides for RSV N-RNA and Influence of Peptide Length on Fluorescent Label Mobility
2.5. Fluorescein Binds to the RSV P-Binding Site on RSV N Protein
2.6. An FP Assay for the RSV N-P Interaction Using Full-Length N and BODIPY FL-Labeled P Peptides
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Synthesis of Fluorescent Molecules
4.3. Peptide Synthesis
4.3.1. Procedure A: Fmoc Removal
4.3.2. Procedure B: Coupling Steps
4.3.3. Procedure C: Coupling of Dicyano BODIPY FL
4.3.4. Procedure D: Resin Cleavage and Protecting Groups Removal
4.4. Peptide Purification and Yields
4.4.1. Unlabeled Peptides
- P3 peptide was obtained as a white powder (34.0 mg, 50%). Analytical UPLC tr = 0.87 min; ESI-MS (positive mode) calculated for [C18H23N3O8], 409.2; found m/z, 410.4 (M + H)+. [Focused gradient 5–25% of B in 15 min].
- P4 peptide was obtained as a white powder (49.0 mg, 59%). Analytical UPLC tr = 1.01 min; ESI-MS (positive mode) calculated for [C24H34N4O9], 522.2; found m/z, 523.5 (M + H)+. [Focused gradient 10–30% of B in 15 min].
- P5E peptide was obtained as a white powder (59.0 mg, 59%). Analytical UPLC tr = 1.00 min; ESI-MS (positive mode) calculated for [C29H41N5O12], 651.3; found m/z, 652.6 (M + H)+. [Focused gradient 15–35% of B in 15 min].
- P7E peptide was obtained as a white powder (43.0 mg, 33%). Analytical UPLC tr = 1.43 min; ESI-MS (positive mode) calculated for [C39H57N7O16], 879.4; found m/z, 880.8 (M + H)+. [Focused gradient 25–35% of B in 15 min].
- P9E peptide was obtained as a white powder (27.0 mg, 17%). Analytical UPLC tr = 1.29 min; ESI-MS (positive mode) calculated for [C47H68N10O21], 1108.5; found m/z, 1109.7 (M + H)+. [Focused gradient 20–30% of B in 15 min].
- P11EE: SPPS of P11EE peptide was performed on 150 mg of 2-CTC resin. The peptide P11EE was obtained as a white powder (28.0 mg, 20%). Analytical UPLC tr = 1.28 min; ESI-MS (positive mode) calculated for [C56H80N12O27], 1352.5; found m/z, 1353.9 (M + H)+. [Focused gradient 20–30% of B in 15 min].
- P11SE: SPPS of P11SE peptide was performed on 15.0 mg of 2-CTC resin. P11SE was obtained as a white powder (4.50 mg) and was used without further purification for F11SE synthesis.
4.4.2. Dicyano BODIPY FL-Labeled BCNn Peptides
- BCN5E: Starting from P5E on resin (8.71 µmol), BCN5E was obtained as a red powder (2.63 mg, 32%). Analytical UPLC tr = 1.61 min; ESI-MS (negative mode) calculated for [C45H54BN9O13], 939.8; found m/z, 939.4 (M − H)−. [Focused gradient 38–48% of B in 15 min].
- BCN6E: Starting from P6E on resin (8.71 µmol), BCN6E was obtained as a red powder (1.60 mg, 18%). Analytical UPLC tr = 1.74 min; ESI-MS (negative mode) calculated for [C51H65BN10O14], 1052.5; found m/z, 1051.2 (M − H)−. [Focused gradient 43–53% of B in 15 min].
- BCN7E: Starting from P7E on resin (8.71 µmol), BCN7E was obtained as a red powder (2.22 mg, 22%). Analytical UPLC tr = 1.66 min; ESI-MS (negative mode) calculated for [C55H70BN11O17], 1167.5; found m/z, 1166.9 (M − H)−. [Focused gradient 40–50% of B in 15 min].
- BCN8E: Starting from P8E on resin (8.71 µmol), BCN8E was obtained as a red powder (2.00 mg, 18%). Analytical UPLC tr = 1.56 min; ESI-MS (positive mode) calculated for [C59H76BN13O19], 1282.6; found m/z, 1283.9 (M + H)+. [Focused gradient 36–46% of B in 15 min].
- BCN9E: Starting from P9E on resin (8.71 µmol), B9E was obtained as a red powder (1.17 mg, 10%). Analytical UPLC tr = 1.53 min; ESI-MS (positive mode) calculated for [C63H81BN14O22], 1396.6; found m/z, 1398.2 (M + H)+. [Focused gradient 35–45% of B in 15 min].
- BCN10EE: Starting from P10EE on resin (8.71 µmol), BCN10EE was obtained as a red powder (1.00 mg, 8%). Analytical UPLC tr = 1.50 min; ESI-MS (negative mode) calculated for [C68H88BN15O25], 1525.6; found m/z, 1525,1(M − H)−. [Focused gradient 34–44% of B in 15 min]. UV-Visible absorption spectrum and fluorescence excitation/emission spectra are given in Figure S33.
- BCN11EE: Starting from P11EE on resin (8.71 µmol), BCN11EE was obtained as a red powder (1.01 mg, 11%). Analytical UPLC tr = 1.49 min; ESI-MS (negative mode) calculated for [C72H93BN16O28], 1640.6; found m/z, 1640.3 (M − H)−. [Focused gradient 33–43% of B in 15 min].
- BCN11SE: Starting from P11SE on resin (4.35 µmol), BCN11SE was obtained as a red powder (1.12 mg, 16%). Analytical UPLC tr = 1.48 min; ESI-MS (negative mode) calculated for [C70H91BN16O27], 1598.6; found m/z, 1598.2 (M − H)−. [Focused gradient 32–42% of B in 15 min].
4.4.3. Fluorescein-Labeled Fn-Peptides
- F3: Starting from P3 (5.00 mg, 9.55 µmol), F3 was obtained as a yellow powder (3.00 mg, 48%). Analytical UPLC tr = 1.31 min; ESI-MS (negative mode) calculated for [C39H34N4O13S], 798.2; found m/z, 797.6 (M − H)−. [Focused gradient 25–45% of B in 15 min].
- F4: Starting from P4 (5.00 mg, 7.85 µmol), F4 was obtained as a yellow powder (4.30 mg, 62%). Analytical UPLC tr = 2.12 min; ESI-MS (negative mode) calculated for [C45H45N5O14S], 911.3; found m/z, 910.6 (M − H)−. [Focused gradient 55–65% of solvent D consisted of MeOH containing 9.9% (v/v) H2O and 0.1% TFA in 15 min].
- F5E: Starting from P5E (5.00 mg, 6.53 µmol), F5E was obtained as a yellow powder (3.60 mg, 53%). Analytical UPLC tr = 0.98 min; ESI-MS (positive mode) calculated for [C50H52N6O17S], 1040.3; found m/z, 1041.8 (M + H)+. [Focused gradient 35–45% of B in 15 min].
- F7E: Starting from P7E (5.00 mg, 5.03 µmol), F7E was obtained as a yellow powder (2.60 mg, 50%). Analytical UPLC tr = 1.53 min; ESI-MS (positive mode) calculated for [C60H68N8O21S], 1268.4; found m/z, 1270.0 (M + H)+. [Focused gradient 30–50% of B in 15 min].
- F9E: Starting from P9E (5.00 mg, 4.09 µmol), F9E was obtained as a yellow powder (1.40 mg, 22%) Analytical UPLC tr = 1.42 min; ESI-MS (positive mode) calculated for [C68H79N11O26S], 1497.5; found m/z, 1499.7 (M + H)+. [Focused gradient 30–50% of B in 15 min].
- F11: Starting from commercial P11 (4.30 mg, 3.11 µmol), F11SS was obtained as a yellow powder (3.5 mg, 68%). Analytical UPLC tr = 1.45 min; ESI-MS (positive mode) calculated for [C73H87N13O30S], 1657.5; found m/z, 1659.8 (M + H)+. [Focused gradient 25–45% of B in 15 min].
- F11SE: Starting from P11SE (4.50 mg, 3.43 µmol), F11SE was obtained as a yellow powder (0.90 mg, 15%). Analytical UPLC tr = 1.39 min; ESI-MS (negative mode) calculated for [C75H89N13O31S], 1699.6; found m/z, 1697.7 (M − H)−. [Focused gradient 25–45% of B in 15 min].
- F11EE: Starting from P11EE (5 mg, 3.41 µmol), F11EE was obtained as a yellow powder (2.40 mg, 40%). Analytical UPLC tr = 1.39 min; ESI-MS (positive mode) calculated for [C77H91N13O32S], 1741.6; found m/z, 1742.6 (M + H)+. [Focused gradient 25–45% of B in 15 min].
- pF11: Starting from commercial pP11 (4.20 mg, 2.93 µmol), pF11 was obtained as a yellow powder (1.10 mg, 20%). Analytical UPLC tr = 1.36 min; ESI-MS (negative mode) calculated for [C73H89N13O36P2S], 1817.5; found m/z, 908.7 (M − 2H)2−. [Focused gradient 25–45% of B in 15 min].
4.5. Bacterial Expression and Purification of RSV N Protein
4.6. NMR Measurements
4.7. Fluorescence Polarization Measurements
4.8. Complex Modelling with Haddock
4.9. Docking of Small Compounds
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fn Peptide | Peptide Sequence | Kd (µM) | ΔFPmax (mFP) |
---|---|---|---|
F11 | ϕ-DS232DNDLS237LEDF-OH | 7.6 ± 0.9 | 130 ± 5 |
F11EE | ϕ-DE232DNDLE237LEDF-OH | 0.38 ± 0.12/0.15 ± 0.12 (*) | 114 ± 6/93 ±13 (*) |
F11SE | ϕ-DS232DNDLE237LEDF-OH | 0.87 ± 0.26/0.62 ± 0.25 (*) | 111 ± 9/97 ± 12 (*) |
F9E | ϕ-DNDLE237LEDF-OH | 2.2 ± 0.3 | 148 ± 5 |
F7E | ϕ-DLE237LEDF-OH | 2.5 ± 0.3 | 187 ± 7 |
F5E | ϕ-E237LEDF-OH | 5.9 ± 0.5 | 285 ± 7 |
F4 | ϕ-LEDF-OH | 35 ± 4 | 291 ± 20 |
F3 | ϕ-EDF-OH | 62 ± 19 | 315 ± 61 |
Fn Peptide | Inhibitor | IC50 (µM) | Ki (µM) | Kd of Fn Peptide (µM) * | Kd of Fn Equivalent to Inhibitor (µM) * |
---|---|---|---|---|---|
F11 | P11EE | 22 ± 4 | 6 | 7.6 | 0.38/0.15 |
F11 | P9E | 66 ± 6 | 25 | 7.6 | 2.2 |
F11 | P7E | 180 ± 11 | 75 | 7.6 | 2.5 |
F11 | P11 | 86 ± 7 | 34 | 7.6 | 7.6 |
F7E | P7E | 376 ± 26 | 74 | 2.5 | 2.5 |
F5E | P5E | 433 ± 21 | 158 | 5.9 | 5.9 |
F11 | M76 | 13 ± 2 | 3 | 7.6 | |
F7E | M76 | 48 ± 5 | 8 | 2.5 |
Fluorescent Molecule | Kd (µM) | M76 IC50 (µM) | M76 Ki (µM) |
---|---|---|---|
Fluorescein 1 | 91 ± 5 | 27 ± 2 | 13 |
Fluorescein methyl ester 2 | 200 ± 8 | 46 ± 7 | 32 |
Fluorescein ethyl ester 3 | 250 ± 27 | 65 ± 9 | 50 |
4,4′ difluoro BODIPY FL 4 | 870 ± 140 | (*) | (*) |
Fluorescent BCNn Peptide | Fluorescent Peptide Sequence | ΔFPmax (mFP) | Kd (µM) | ΔFPmax (mFP) | Kd (µM) |
---|---|---|---|---|---|
No Salt | No Salt | With Salt | With Salt | ||
BCN11SE | BCN-DSDNDLELEDF-OH | 312 ± 25 | 0.82 ± 0.22 | 122 ± 2 | 0.55 ± 0.07 |
BCN11EE | BCN-DEDNDLELEDF-OH | 316 ± 12 | 0.28 ± 0.07 | 197 ± 5 | 0.26 ± 0.04 |
BCN10EE | BCN-EDNDLELEDF-OH | 270 ± 7 | 0.16 ± 0.03 | 171 ± 16 | 1.24 ± 0.31 |
BCN9E | BCN-DNDLELEDF-OH | 288 ± 15 | 0.96 ± 0.13 | ||
BCN8E | BCN-NDLELEDF-OH | 298 ± 5 | 1.43 ± 0.08 | 284 ± 15 (*) | 7.1 ± 0.1 (*) |
BCN7E | BCN-DLELEDF-OH | 332 ± 8 | 2.2 ± 0.2 | ||
BCN6E | BCN-LELEDF-OH | 275 ± 8 | 2.3 ± 0.2 | 281 ± 19 (*) | 12.6 ± 1.6 (*) |
BCN5E | BCN-ELEDF-OH | 431 ± 15 | 9.8 ± 0.9 |
Fluorescent Peptide | Inhibitor | [N-RNA] (µM) | Fitted FPmax (mFP) | Fitted IC50 (µM) | Ki (µM) | Kd (µM) of BCN-P Peptide (*) |
---|---|---|---|---|---|---|
BCN10EE | P10EE | 1 | 214 | 21 ± 4 | 2.9 | 0.16 |
BCN11EE | pP11 | 1 | 234 | 6.9 ± 1.2 | 1.4 | 0.28 |
BCN11SE | M76 | 5 | 280 | 17 ± 2 | 1.8 | 0.8 |
BCN10EE | M76 | 2 | 257 | 8.9 ± 0.6 | 0.5 | 0.16 |
BCN10EE | M76 | 1 | 219 | 8.5 ± 0.6 | 1.1 | 0.16 |
BCN8E | M76 | 5 | 196 | 13 ± 2 | 2.0 | 1.4 |
BCN8E | M76 | 2 | 166 | 4.5 ± 0.4 | 1.3 | 1.4 |
BCN6E | M76 | 10 | 177 | 14 ± 1 | 1.1 | 2.3 |
BCN6E | M76 | 2 | 104 | 2.7 ± 0.2 | 0.7 | 2.3 |
BCN5E | M76 | 20 | 280 | 26 ± 2 | 3.6 | 9.8 |
BCN5E | M76 | 2 | 76 | 7.0 ± 0.6 | 4.8 | 9.8 |
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Khodjoyan, S.; Morissette, D.; Hontonnou, F.; Checa Ruano, L.; Richard, C.-A.; Sperandio, O.; Eléouët, J.-F.; Galloux, M.; Durand, P.; Deville-Foillard, S.; et al. Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization. Int. J. Mol. Sci. 2023, 24, 569. https://doi.org/10.3390/ijms24010569
Khodjoyan S, Morissette D, Hontonnou F, Checa Ruano L, Richard C-A, Sperandio O, Eléouët J-F, Galloux M, Durand P, Deville-Foillard S, et al. Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization. International Journal of Molecular Sciences. 2023; 24(1):569. https://doi.org/10.3390/ijms24010569
Chicago/Turabian StyleKhodjoyan, Silva, Deborha Morissette, Fortune Hontonnou, Luis Checa Ruano, Charles-Adrien Richard, Olivier Sperandio, Jean-François Eléouët, Marie Galloux, Philippe Durand, Stéphanie Deville-Foillard, and et al. 2023. "Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization" International Journal of Molecular Sciences 24, no. 1: 569. https://doi.org/10.3390/ijms24010569